The demand for synthetic oligonucleotides has grown exponentially as genome sequencing, functional genomics, and PCR-based detection methods consume enormous quantities of DNA oligonucleotides. In addition, the potential success of new DNA- and RNA-based therapeutic platforms (such as antisense and siRNA gene silencing strategies) currently undergoing clinical trials could result in an unprecedented demand for short synthetic DNA and RNA molecules.
RNA interference (RNAi) as potential therapeutics represents a fundamentally new way to treat human diseases [Manoharan, Curr. Opin. Chem. Biol. 8, 570-579 (2004)]. However, achieving targeted tissue and cellular delivery, stabilization in vivo, and cost effective large scale synthesis of RNA are significant bottlenecks in the development of RNAi technology. The reality of mainstream RNAi based therapeutics is rapidly approaching and the demand for these compounds on large scale may soon exceed the capability of manufacturers. Therefore, there is a need to develop synthetic strategies enabling RNA oligomers to be synthesized rapidly, in high purity and/or in a cost efficient, large scale synthesis.
Current methods for DNA and RNA synthesis rely on stepwise addition of monomeric phosphoramidite units on solid supports [Caruthers, M. H. et al. Methods in Enzymology 154, 287-313 (1987); Alvarado-Urbina, G. et al. Science 214, 270-274 (1981)]. Chain elongation from 3′- to 5′-end is preferred, which is achieved by coupling of a nucleoside unit having a 3′-phosphorus (III) group (in its activated form) to a free 5′-hydroxyl group of another nucleoside unit. As solid support, 500 to 1000 Å controlled pore glass (CPG) support or an organic polymer support, such as primer polystyrene support PS200, can be used. Chain elongation begins by cleavage of the 5′-O-dimethoxytrityl group with an organic acid, thus liberating a nucleophilic 5′-hydroxyl group. This terminal nucleophile is then allowed to couple to a protected nucleoside 3′-O-phosphoramidite monomer in the presence of an activator. In the case of RNA synthesis suitable protection of the 2′-hydroxyl group is required. Any unreacted 5′-hydroxyl groups are acetylated in a process referred to as ‘capping’. The most commonly used group used for this purpose is an acetyl ester. Thus, ‘capping’ with acetic anhydride esterifies any unreacted 5′-hydroxyl groups and halts the accumulation of by-products. The newly created phosphite triester 3′,5′-linkage is then oxidized to provide the desired and more stable phosphate triester. This process is repeated until an oligomer of the desired length and sequence is obtained. Cleavage of the oligomer from the solid support and removal of the protecting groups from the sugars, phosphates and nucleobases provides the desired target oligomer, which is then separated from shorter failure sequences by ion exchange high pressure liquid chromatography (HPLC), ion-pair reverse phase HPLC, or polyacrylamide gel electrophoresis (PAGE). The full length oligomer is then characterized by mass spectrometry. Meanwhile a large number of DNA oligomers can be synthesized in parallel on DNA microarrays or “gene chips” [Ramsay G., Nature Biotechnology 16, 40-44 (1998)].
The same iterative method may be applied toward the synthesis of DNA and RNA oligonucleotides in solution, for example as described recently by Donga et al. using ionic soluble supports [e.g. Donga, R. A. et al., J. Org. Chem. 71, 7907-7910 (2006); Donga, R. A. et al., Can. J. Chem. 85, 274-282 (2007)]. The use of ionic soluble supports allows for selective precipitations of the growing oligonucleotide over all other reagents used in the oligonucleotide synthesis cycle.
To date, there have been many attempts to design protecting groups and methods that embody the conditions required for the construction of high quality oligoribonucleotides [for reviews, see Beaucage, S. L. Curr. Opin. Drug Discov. Devel. 11, 203-16 (2008); Reese, C. B. Organic & Biomolecular Chemistry 3, 3851-3868 (2005)]. In fact, for many years, RNA synthesis has been regarded as far more complicated than DNA synthesis because of the difficulty in finding a compatible 2′-protecting group that (a) affords high step-wise coupling yields, (b) is stable throughout chain assembly, and (c) can be removed selectively at the end of synthesis without phosphodiester bond isomerization or degradation.
The most widely used 2′-protecting group is the 2′-O-t-butyldimethylsilyl (TBDMS) group [Ogilvie, K. K. et al. Tetrahedron Letters 15, 2861-2867 (1974)]. This protecting group is removed at the end of RNA chain assembly in the presence of fluoride ions. Other silyl ether based protecting groups described for the protection of nucleosides are triisopropylsilyl (TIPS), methyldiisopropylsilyl (MDIPS), cyclic alkylsilyl and other silyl groups [Ogilvie, K. K. et al. J. of Carbohydrates, Nucleosides, Nucleotides 3, 197-227 (1976); Damha M. J, Ogilvie K. K. (1993), Oligoribonucleotide synthesis: the silyl-phosphoramidite method. In: Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology (Agrawal S, ed.) Vol. 20. Totowa, N.J.: The Humana Press Inc. pp. 81-114]. Among these, TIPS protection has been described primarily for 5′-O-monomethoxytrityl N2-isobutyrylguanosine derivatives as the 2′ and 3′-O-TIPS isomers are more readily separated from each other by silica gel chromatography [Damha M. J, Ogilvie K. K. (1993), Oligoribonucleotide synthesis: the silyl-phosphoramidite method. In: Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology (Agrawal S, ed.) Vol. 20. Totowa, N.J.: The Humana Press Inc. pp. 81-114; Damha, M. J. et al. Tetrahedron Letters 45, 6739-6742 (1992)]. The smaller steric bulk of the TBDMS group relative to TIPS, TBDPS and other bulkier silyl ethers would favor the TBDMS protecting group, which has been used the most compared to other protecting group for RNA synthesis. Coupled with the phosphoramidite condensation-procedure, 2′-O-TBDMS monomers have allowed a highly efficient synthesis of oligoribonucleotides [Ogilvie, K. K. et al. Proc. Natl. Acad. Science (USA), 85, 5764-5768 (1988); Usman, N. et al. J. Am. Chem. Soc. 109, 7845-7854 (1987)].
A potential drawback of silyl ethers for the protection of the 2′-hydroxyl group lies in their widely recognized ability to undergo 2′-to-3′ isomerization under the influence of protic solvents, nucleophilic catalysts, or basic conditions. For example, the TBDMS group migrates from the 02′ to 03′ position (and vice versa) in the presence of either methanol, imidazole, pyridine/water, or aqueous ammonia, thereby generating a mixture of nucleoside O2′ and O3′ silyl regioisomers [Ogilvie, K. K. and Entwistle, D. W. Carbohydrate Res. 89, 203-210 (1981); Ogilvie, K. K. (1983) Proceedings of the 5th International Round Table on Nucleosides, Nucleotides and Their Biological Applications, (Rideout, J. L. et al. eds.), Academic, London, pp. 209-256); Damha M. J, Ogilvie K. K. (1993), Oligoribonucleotide synthesis: the silyl-phosphoramidite method. In: Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology (Agrawal S, ed.) Vol. 20. Totowa, N.J.: The Humana Press Inc. pp. 81-114].
Silyl isomerization is characteristic of other 0-silyl ether protecting groups. 0-TIPS derivatives of uridine and 7-deazaguanosine also undergo isomerization in methanol, albeit more slowly than their O-TBDMS counterparts [Ogilvie, K. K. et al. J. or Carbohydrates, Nucleosides, Nucleotides 3, 197-227 (1976); Seela, F. and Mersmann, K., Helvetica Chimica Acta, 76, 1435-1449(1993)]. 5′-O-Monomethoxytrityl-N2-isobutyryl-2′-O-TIPS guanosine undergoes isomerization under ethanolic aq. ammonia conditions to give a mixture of 2′/3′-TIPS regioisomers which can be separated by chromatography. This provides a method to convert (recycle) more of the unwanted 3′-isomer into the more useful 2′-isomer [Damha M. J, Ogilvie K. K. (1993), Oligoribonucleotide synthesis: the silyl-phosphoramidite method. In: Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology (Agrawal S, ed.) Vol. 20. Totowa, N.J.: The Humana Press Inc. pp. 81-114].
2′-O-Silyl groups do not normally migrate to O3′ in dry aprotic solvent. When these conditions are strictly adhered to it is possible to prepare 2′-O-silylated ribonucleoside-3′-O-phosphoramidite derivatives in regiosomerically pure form [Milecki, J. et al. Nucleosides & Nucleotides, 8, 463-474 (1989); Scaringe, S. A. et al. Nucleic Acids Res., 18, 5433-5341 (1990)]. This is clearly an important requirement as the presence of even traces of the 3′-O-silyl regioisomer will impact on the quality and biological activity of the desired RNA sequence.
Many other protecting groups for the 2′-hydroxyl position have been used in the synthesis of RNA [reviewed in Beaucage, S. L. Curr. Opin. Drug Discov. Devel. 11, 203-16 (2008); Reese, C. B. Organic & Biomolecular Chemistry 3, 3851-3868 (2005)]. RNA synthesis using monomers containing the 2′-triisopropylsilyloxymethyl (TOM) group, the 2′-acetal-levulinyl group, and the 2′-O-bis(2-acetoxyethoxy)methyl (ACE) group, have been reported to yield higher coupling efficiency, because these protecting groups exhibit lower steric hindrance than the 2′-TBDMS group [for a comparative study, see Lackey, J. G. et al. J. Am. Chem. Soc. 131, 8496-8502 (2009)]. Like the TBDMS group, the TOM protecting group is removed using fluoride.
In all cases the synthesis of oligoribonucleotides is an elaborate multistep process, which entails assembly of the oligonucleotide chain typically from monomeric phosphoramidite building blocks (e.g., 5′-O-dimethoxytrityl-N-protected-2′-O-tert-butyldimethylsilyl-nucleoside-3′-O-phosphoramidites), deprotection of the base labile nucleobase protecting groups (e.g., benzoyl, isobutyryl, acetyl, phenoxyacetyl, levulinyl, etc), cleavage from the support (e.g., glass beads or polystyrene), followed by removal of the 2′-hydroxyl protecting group.
The generation of oligoribonucleotide blocks is more difficult due to the presence of the 2′-hydroxyl group and the protection it requires, thus this line of research has also lagged far behind that of DNA blocks. Nevertheless, there have been several reports describing the synthesis of RNA through block coupling condensation reactions. Ikehara and co-workers coupled RNA trimer and tetramers using the phosphotriester method to give 30% yield after several days [Ohtsuka, E. et al. J. Am. Chem. Soc. 100, 8210 (1978)]. Werstiuk and Nielson reported the coupling of an RNA tetramer and an RNA pentamer affording the desired nonanucleotide RNA sequence in 50% yield after 16 days [Werstiuk, E. S., Neilson, T. Can. J. Chem. 54, 2689 (1976)]. Van Boom and co-workers condensed an RNA tetramer and an RNA decamer in 58% yield in a 3.5 days reaction [van Boom, J. H. et al. Trav. Chim. Pays-Bas, 97, 73 (1978)]. Ogilvie and co-workers described the synthesis of 5′-O-monomethoxytrityl-2′-O-tert-butyldimethylsilyl-3′-O-levulinyl ribonucleoside monomers and their use in the assembly of a hexadecauridylic acid via the phosphodichloridite procedure [Nemer, M. J, and Ogilvie, K. K., Can. J. Chem. 58, 1389-1397 (1980)].
Solid-phase RNA synthesis is carried out almost exclusively using monomeric phosphoramidite synthons. Given the efficiency of the phosphoramidite chemistry, it is highly desirable to have access to block (dimer and trimer) phosphoramidites for RNA synthesis, as these would permit longer chain extensions at each step during chain assembly, significantly shortening the time required for synthesis.
However, while solid-supported synthesis overcomes the limitation of purification by allowing excess reagents to be washed away, it can be quite restricting in terms of scale. While it is true that current large scale methods of producing oligonucleotides in the kilogram scale utilize solid phase approaches, the mechanical requirements for this type of manufacturing are very specialized and costly. Therefore, an ideal method of large scale synthesis is in solution. In attempts to overcome this limitation, a variety of soluble polymer-based supports have been developed [Gravert, D. J., Janda, K. D. Chem. Rev. 97, 489-509 (1997)]. These however suffer from their own limitations such as poor loading, unfavorable atom economy, and the reliance on temperature cycling to solvate/precipitate the soluble support. Some unique perfluorinated “supports” have been reported that are covalently attached to a desired molecule and hence adhere to long chain fluorocarbon derivatized silica. They can be selectively removed by perfluorinated solvents. This is a very efficient process, but requires many specialized and expensive materials [Horvath, I. T., Rabai, J. Science, 266, 72-75. (1994); Studer, A. et al., Science, 275, 823-826, (1997)].
It is also desirable therefore to have improved methods for solution phase RNA synthesis.